A new subfamily of cytokines capable of regulating bone cell function has been identified. The members of this LIF/IL-6 subfamily include leukemia inhibitory factor, oncostatin M, interleukin-6, interleukin-11, ciliary neurotrophic factor, and cardiotrophin-1. Because of their potent regulatory activity on bone cells, these factors have been implicated in the regulation of normal bone remodeling and the etiology of bone diseases like osteoporosis. It is the application's hypothesis that members of this subfamily activate osteoblasts through specific receptors, resulting in tyrosine phosphorylation of a specific set of proteins. Some of these proteins are transcription factors that, once phosphorylated, translocate to the nucleus and induce gene activation, while others are involved in regulation of the signal transduction pathway itself. Gene activation results in the transcription of proteins which regulate bone cell function. The long-term goal of this proposal is to elucidate the signal transduction pathway from the cell surface to the nucleus, and identify the biological outcome induced by these cytokines using biochemical, molecular and functional techniques. It is suggested that four distinct, but interrelated Specific Aims will be pursued: 1) to identify the cytokine subfamily members which activate osteoblasts; 2) to identify the tyrosine phosphorylated proteins induced by the cytokines in osteoblasts; 3) to characterize the genetic and biologic effects of ligand binding; and 4) to analyze the osteopenia in LIFR knock-out mice. Murine osteoblasts will be compared with human osteoblast-like cells for activation by the cytokines. Activation will be determined by induction of tyrosine phosphorylated proteins and cytokine secretion. Tyrosine phosphorylated proteins will be identified by immunoprecipitation and Western blotting. Genes and gene products activated by ligand binding will be identified by Northern blot and functional analysis. The LIFR component of the receptor used by some of these cytokines was deleted by homologous recombination. These mice develop multiple skeletal abnormalities, including severe osteopenia with increased numbers of osteoclasts. Histomorphometry and cell mixing experiments with mutant and control osteoblasts and osteoclast precursors will be used to analyze the bone defect. The applicant suggests that elucidation of the components of the signal transduction pathway used by these factors will lead to a clearer understanding of how normal bone cell function is regulated, and the development of bone diseases such as osteoporosis. Once understood, these results might then be applied to develop better approaches and targets for therapy.